Antibodies have proven to be very effective and successful therapeutic agents in the treatment of cancer, autoimmune diseases and other disorders. While full-length antibodies typically have been used clinically, there are a number of advantages that use of an antibody fragment can provide, such as increased tissue penetration, absence of Fc-effector function combined with the ability to add other effector functions and the likelihood of less systemic side effects resulting from a shorter in vivo half life systemically. The pharmacokinetic properties of antibody fragments indicate that they may be particularly well suited for local therapeutic approaches. Furthermore, antibody fragments can be easier to produce than full-length antibodies in certain expression systems.
One type of antibody fragment is a single chain antibody (scFv), which is composed of a heavy chain variable domain (VH) conjugated to a light chain variable domain (VL) via a linker sequence. Thus, scFvs lack all antibody constant region domains and the amino acid residues of the former variable/constant domain interface (interfacial residues) become solvent exposed. An scFv can be prepared from a full-length antibody (e.g., IgG molecule) through established recombinant engineering techniques. The transformation of a full length antibody into an scFv, however, often results in poor stability and solubility of the protein, low production yields and a high tendency to aggregate, which raises the risk of immunogenicity.
Accordingly, attempts have been made to improve properties such as solubility of scFvs. For example, Nieba, L. et al. (Prot. Eng. (1997) 10: 435-444) selected three amino acid residues known to be interfacial residues and mutated them. They observed increased periplasmic expression of the mutated scFv in bacteria, as well as a decreased rate of thermally induced aggregation, although thermodynamic stability and solubility were not significantly altered. Moreover, in their publication, they expressively state they did not observe any solubility improvement of the native protein state of the engineered scFvs as determined by the PEG precipitation method. Other studies in which site directed mutagenesis was carried out on particular amino acid residues within the scFv also have been reported (see e.g., Tan, P. H. et al. (1988) Biophys. J. 75: 1473-1482; Worn, A. and Pluckthun, A. (1998) Biochem. 37: 13120-13127; Worn, A. and Pluckthun, A. (1999) Biochem. 38: 8739-8750). In these various studies, the amino acid residues selected for mutagenesis were chosen based on their known positions within the scFv structure (e.g., from molecular modeling studies).
In another approach, the complementary determining regions (CDRs) from a very poorly expressed scFv were grafted into the framework regions of an scFv that had been demonstrated to have favorable properties (Jung, S. and Pluckthun, A. (1997) Prot. Eng. 10: 959-966). The resultant scFv showed improved soluble expression and thermodynamic stability.
Progress in the engineering of scFvs to improve solubility and other functional properties is reviewed in, for example, Worn, A. and Pluckthun, A. (2001) J. Mol. Biol. 305: 989-1010. New approaches, however, are still needed that allow for rational design of immunobinders, in particular of scFvs with superior solubility. Moreover, methods of engineering scFvs, and other types of antibodies, to thereby impart improved solubility—especially solubility of the native protein—, are still needed.